Application of substance for improving LSH content or activity in improving heterochromatin stability and enhancing cell treatment effect

Abstract

The invention discloses an application of a substance for improving the content or activity of LSH in improving the stability of heterochromatin and enhancing the therapeutic effect of cells, and belongs to the technical field of biological medicines. Complete inhibition of DNA methylation loss in the heterochromatin region in the cell amplification process is successfully realized for the first time. The innovative research result of the invention powerfully proves that the insufficient DNA methylation maintenance efficiency caused by a dense chromatin environment in a heterochromatin region is a reason for continuous loss of DNA methylation during continuous cell amplification. And overexpression of the LSH can effectively improve the DNA methylation maintenance efficiency of the heterochromatin region to prevent DNA methylation loss, so that multiple phenotype defects caused by continuous cell amplification are improved. A new strategy is provided for effectively maintaining the cell function in cell adoptive therapy, and the method has wide application value in the aspect of improving the therapeutic efficacy of various cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a substance that increases the content or activity of LSH in improving the stability of heterochromatin and enhancing the therapeutic effect of cell therapy. Background Art

[0002] Cell therapy is a method of delivering autologous or allogeneic living cells with normal or enhanced functions into the patient's body as therapeutic drugs to repair, replace or eliminate damaged or abnormal cells in the body, thereby achieving tissue regeneration or lesion removal. Currently, the most widely used cell drugs in the field of cell therapy mainly include stem cell transplantation therapy (such as hematopoietic stem cell transplantation and mesenchymal stem cell transplantation) and immune cell adoptive therapy (such as CAR-T therapy and TCR-T therapy). As a new type of treatment, these cell therapy drugs have shown great application value in the treatment of various complex diseases (such as autoimmune diseases, neurodegenerative diseases and tumors) compared with traditional chemical small molecules or biological macromolecules.

[0003] Although cell therapy has achieved widespread success, it still encounters many unresolved problems in its practical application. Among them, insufficient cell proliferation ability and poor persistence after in vivo transplantation are one of the main problems encountered by various cell therapy methods. For example, mesenchymal stem cell transplantation therapy first requires the preparation of a sufficient number of clinical-grade mesenchymal stem cells in vitro, and long-term cell subculture in this process will induce cell senescence, which is mainly manifested in decreased cell proliferation ability, increased chromosomal abnormalities, and increased inflammatory response. Similarly, in immune cell adoptive therapy (such as CAR-T therapy), it is first necessary to amplify a large number of autologous T lymphocytes from patients in vitro, and the poor in vitro proliferation ability of T lymphocytes from some patients is the main limiting factor for the inability to obtain a sufficient number of CAR-T cells for in vivo transfusion, and low-dose CAR-T cells cannot completely and effectively eliminate tumor cells. At the same time, CAR-T therapy still has bottlenecks in the treatment of solid tumors, among which the tumor microenvironment of solid tumors poses a challenge to the persistent maintenance of the proliferation ability and effector function of CAR-T cells. After being infused back into the patient's body, CAR-T cells often show exhausted or aged cell phenotypes after entering the tumor microenvironment, making it impossible to effectively and continuously eliminate tumor cells in the long term. Therefore, how to effectively improve the proliferation ability of cells in vitro or in vivo and prevent cell function degradation during cell therapy is one of the main problems encountered by various cell drugs in the application of diseases.

[0004] Epigenetic information imbalance is one of the important factors leading to cell function degradation. Among them, the loss of DNA methylation in heterochromatin regions caused by massive cell proliferation is an important epigenetic feature shared by aging and tumor cells. This chromatin change can also be seen in a variety of cell drug treatments, such as cell senescence during in vitro expansion of mesenchymal stem cells, and exhaustion or aging of CAR-T cells in the tumor microenvironment after they are reinfused into the patient's body. Significant reductions in heterochromatin-specific DNA methylation levels can be seen. As the main mechanism responsible for transcriptional silencing in heterochromatin regions in somatic cells, a decrease in DNA methylation levels can lead to abnormal activation of a large number of repetitive sequences or transposon elements in heterochromatin regions, which in turn triggers the activation of intracellular nucleic acid sensing pathways (such as cGAS-STING pathways, etc.), thereby inducing the generation of chronic inflammatory responses and impairing the maintenance of normal cell functions. At the same time, heterochromatin activation in centromere / paracentromere regions will also undermine the maintenance of genomic stability, thereby affecting the normal chromosome separation process, leading to defects in cell division ability.

[0005] Although heterochromatin DNA methylation plays a key role in preventing abnormal activation of transposons, maintaining genome stability, and reducing chronic inflammatory responses, the reason for the continuous loss of heterochromatin DNA methylation during long-term cell proliferation is still unclear, and there is a lack of effective intervention measures to prevent the loss of heterochromatin DNA methylation. In a previous study, the applicant systematically compared the maintenance rate of DNA methylation in different regions of the genome during DNA replication, and found that the dense chromatin environment in the heterochromatin region is not conducive to the effective maintenance of DNA methylation, and this lack of maintenance efficiency may be the reason for the continuous decline in the level of DNA methylation in this region during long-term cell expansion. At the same time, genetic evidence shows that the chromatin remodeling factor LSH is specifically essential for the effective maintenance of DNA methylation in heterochromatin regions. Therefore, we believe that the use of gene overexpression or other methods to increase the content or activity of LSH can effectively inhibit the loss of DNA methylation in heterochromatin regions, thereby improving cell proliferation ability and preventing cell function degradation, thereby enhancing the persistence and therapeutic effect of cell therapy drugs. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a substance that increases the content or activity of LSH (including LSH overexpression recombinant vectors, recombinant viruses and recombinant cells) for use in improving heterochromatin stability and enhancing the efficacy of cell therapy. The substance that increases the content or activity of LSH can effectively prevent the loss of DNA methylation in the heterochromatin region during long-term cell proliferation, thereby reducing the occurrence of chronic inflammatory responses, and significantly improves the cell proliferation ability, while promoting the effective maintenance of cell effector functions, so that it is widely used as an enhancer in various cell therapies.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses for the first time the use of a substance that increases the content / activity of LSH in the preparation of a drug / reagent that increases the stability of heterochromatin.

[0009] Preferably, the substance that increases the expression level of LSH is an LSH overexpression substance that inhibits the loss of DNA methylation in heterochromatin regions.

[0010] Further preferably, the heterochromatin region is a chromatin region highly enriched with histone H3K9me3 modification, HP1 protein or Lamin B1 protein, or a DNA late replication region in the S phase of the cell cycle.

[0011] The invention also discloses the use of a substance for increasing the content / activity of LSH in the preparation of a drug / reagent for enhancing the therapeutic effect of cell therapy.

[0012] Preferably, the drug is a drug that promotes the effective maintenance of cell function during cell therapy.

[0013] further Preferably, the cell therapy includes functional cell transplantation therapy and immune cell adoptive therapy, etc.

[0014] Preferably, the substance that increases the expression level / activity of LSH is a LSH overexpression recombinant vector, recombinant virus or recombinant cell.

[0015] Further preferably, the LSH overexpression recombinant vector is obtained by connecting the LSH gene coding sequence to a viral vector.

[0016] More preferably, the LSH gene coding sequence is the original CDS coding sequence of the LSH gene, or the CDS coding sequence of the LSH gene after codon optimization, or the CDS coding sequence of the LSH gene after base sequence modification without changing the biological function of LSH itself.

[0017] More preferably, the viral vector is a lentiviral vector, a retroviral vector or an AAV viral vector.

[0018] Preferably, the LSH overexpression recombinant lentivirus is produced by co-transfecting the above-mentioned LSH overexpression recombinant vector and a virus packaging plasmid into human embryonic kidney HEK293T cells.

[0019] Further preferably, the virus packaging plasmid is pMDLg / pRRE, pRSV / REV and pVSVG.

[0020] Preferably, the LSH-overexpressing recombinant cells are obtained by infecting host cells with the LSH-overexpressing recombinant virus.

[0021] Further preferably, the host cell is human embryonic lung IMR90 fibroblast, or human peripheral blood-derived T lymphocyte, or other cell types used for cell therapy.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention firstly successfully achieved complete inhibition of DNA methylation loss in heterochromatin regions caused by long-term cell proliferation by overexpressing LSH in a replicative cell senescence model of human embryonic lung IMR90 fibroblasts, thereby effectively preventing abnormal activation of transposon elements, reducing the generation of downstream chronic inflammatory responses, and significantly improving the proliferation capacity of cells, and improving multiple cell senescence-related phenotypes. At the same time, the present invention also effectively alleviated the decrease in DNA methylation levels in heterochromatin regions during clonal expansion by overexpressing LSH in T lymphocytes derived from human peripheral blood, thereby significantly improving the expansion capacity and cell survival of T cells, and promoting the long-term effective maintenance of the tumor killing effector function of T cells.

[0024] Therefore, the innovative research results of the present invention strongly prove that the insufficient efficiency of DNA methylation maintenance caused by the dense chromatin environment of heterochromatin is the reason for the continuous loss of DNA methylation in this region during long-term proliferation, and overexpression of LSH can specifically improve the efficiency of heterochromatin methylation maintenance to prevent the reduction of methylation levels, which provides a new perspective and target for understanding and intervening in the imbalance of epigenetic information that occurs in cells after undergoing a large number of clonal expansions. At the same time, using LSH overexpression to improve heterochromatin stability can significantly improve cell proliferation and survival capabilities, and reduce the occurrence of adverse reactions such as chronic inflammation, thereby promoting the long-term stable maintenance of cell effector functions, which provides a new means to enhance the efficacy of cell therapy drugs, and has shown a wide range of application value in multiple cell adoptive therapies including CAR-T therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1a The transcript level results of HELLS (LSH) gene in IMR90 cells after infection with mCherry and hLSH overexpressing lentivirus obtained by RNA-seq sequencing;

[0026] Figure 1b The protein level of LSH in IMR90 cells infected with mCherry and hLSH overexpressing lentivirus was obtained by Western Blot detection;

[0027] Figure 1c The overall level of DNA methylation in the whole genome of wild-type young IMR90 cells without long-term subculture (P9) and replicative senescent IMR90 cells overexpressing mCherry / hLSH after long-term massive proliferation (P19) obtained by WGBS detection;

[0028] Figure 1d Visualization of DNA methylation levels at specific locations in the genome in wild-type young IMR90 cells and replicative senescent IMR90 cells overexpressing mCherry / hLSH;

[0029] Figure 1e The heat map results are obtained after analyzing the differentially methylated regions (DMRs) with significant changes in DNA methylation in wild-type young IMR90 cells and replicative senescent IMR90 cells overexpressing mCherry / hLSH;

[0030] Figure 1f The DNA methylation levels in the early and late regions of DNA replication in wild-type young IMR90 cells and replicative senescent IMR90 cells overexpressing mCherry / hLSH;

[0031] Figure 2a The volcano plot shows the changes in transposon element expression during replicative senescence of IMR90 cells caused by long-term continuous proliferation;

[0032] Figure 2b is a volcano plot showing the differential expression of transposon elements in replicatively aged IMR90 cells overexpressing mCherry / hLSH;

[0033] Figure 2cTo show the transposon elements that are significantly upregulated during long-term proliferation of IMR90 cells, a heat map of expression changes in IMR90 cells overexpressing mCherry and hLSH, where the transposon elements whose activation can be inhibited by LSH overexpression are defined as Cluster 1, and the transposon elements that cannot be inhibited by LSH overexpression are defined as Cluster 2;

[0034] Figure 2d It shows the changes of DNA methylation on transposon elements whose upregulation can be inhibited by LSH overexpression (Cluster 1) and cannot be inhibited by LSH overexpression (Cluster 2) during long-term proliferation of IMR90 cells;

[0035] Figure 2e The heat map on the left in the middle shows the expression changes of the coding genes in IMR90 cells overexpressing mCherry and hLSH during long-term continuous proliferation, while the right figure shows the genes (Cluster 2) in the left figure that are significantly upregulated during replicative senescence and effectively inhibited by LSH overexpression. The results of biological pathway enrichment analysis (GOterm analysis) using DAVID software, where "*" marks the biological pathways related to inflammatory response;

[0036] Figure 2f Heat map showing the changes of inflammatory response-related genes significantly upregulated during long-term sustained proliferation with replicative senescence in IMR90 cells overexpressing mCherry and hLSH;

[0037] Figure 2g The growth statistics curve of IMR90 cells overexpressing mCherry and hLSH during long-term subculture;

[0038] Figure 2h The changes in cell volume (FSC value) of IMR90 cells overexpressing mCherry and hLSH during long-term subculture were detected by flow cytometry;

[0039] Figure 3a This is a schematic diagram of the process of in vitro separation and purification, antigen activation, virus infection and long-term culture of human peripheral blood T cells;

[0040] Figure 3b This is the change of LSH protein level during long-term expansion of human peripheral blood T cells in vitro;

[0041] Figure 3c The results of Western Blot detection of LSH protein expression in human T cells after infection with mCherry and hLSH lentivirus;

[0042] Figure 3d The changes of DNA methylation in the heterochromatin region (PMD) of the control group cells and LSH-overexpressing cells during the long-term clonal proliferation of T cells detected by WGBS technology;

[0043] Figure 3e The volcano plot shows the changes in the transcriptome of control T cells and LSH-overexpressing T cells after long-term clonal expansion;

[0044] Figure 3f This is the result of GSEA analysis, showing the expression changes of genes related to T cell effector function during long-term clonal expansion and after LSH overexpression;

[0045] Figure 3g This is the statistical curve of the growth of human T cells overexpressing mCherry and hLSH during long-term culture in vitro;

[0046] Figure 3h The results of flow cytometry analysis of Ki67, a cell proliferation-related marker, on the 11th day of in vitro expansion of T lymphocytes in the RFP overexpression control group and the hLSH overexpression experimental group;

[0047] Figure 3i The results of flow cytometry analysis of the change in the proportion of dead cells in late clonal proliferation T cells using the dead cell fluorescent dye A780;

[0048] Figure 3j These are the results of flow cytometric analysis of the cell surface co-stimulatory molecule CD27 during in vitro clonal expansion of T lymphocytes in the RFP overexpression control group and the hLSH overexpression experimental group. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0050] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0051] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0052] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0053] Example 1

[0054] Overexpression of LSH can delay the loss of heterochromatin DNA methylation and replicative senescence phenotype produced in human embryonic lung IMR90 fibroblasts during continuous proliferation.

[0055] The specific operating steps of the experiment involved in this embodiment are as follows:

[0056] 1. Obtaining LSH coding sequence and constructing lentiviral recombinant plasmid

[0057] (1) Obtaining LSH coding sequences

[0058] 1) Wild-type LSH sequence cloning:

[0059] The wild-type gene sequence encoding human LSH was amplified from the cDNA library of human cervical cancer HeLa-S3 cells using nested PCR. The amplification primers are as follows:

[0060] Nested upstream primer 1: 5′-AGCGGTTGTGAGGAGTTAGC-3′ (shown in SEQ ID NO: 1)

[0061] Nested downstream primer 1: 5′-TCTCTCCCCATGAAAAGCCT-3′ (shown in SEQ ID NO: 2)

[0062] Nested upstream primer 2: 5′-CATTGCAGGCTCTGAGAGGA-3′ (shown in SEQ ID NO: 3)

[0063] Nested downstream primer 2: 5′-ACCTAAAGCCCATGAACTGC-3′ (shown in SEQ ID NO: 4)

[0064] After the PCR reaction was completed, the LSH coding sequence fragment of the corresponding size was purified using the gel excision recovery method.

[0065] 2) LSH coding sequence optimization:

[0066] The codon composition of the LSH coding sequence was modified using a codon optimization algorithm, and the optimized LSH coding sequence fragment was obtained through gene synthesis and PCR amplification.

[0067] (2) LSH coding sequence connected to T vector

[0068] The wild-type or optimized LSH coding sequence fragment was connected to the pEASY-T5 cloning vector to construct the pEASY-T5-LSH recombinant plasmid, and the correctness of the obtained LSH coding sequence was verified by transformation of DH5α competent bacteria and single colony Sanger sequencing.

[0069] (3) LSH coding sequence connected to lentiviral expression vector

[0070] The LSH coding sequence and the pLenti lentiviral expression vector were linearized by PCR, and homology arms were introduced at both ends of the fragments. Then, the LSH coding sequence and the pLenti lentiviral expression vector were connected by Gibson Assembly (a molecular biology technique used to assemble DNA molecules in vitro) to construct the pLenti-LSH recombinant plasmid. The correctness of the recombinant plasmid was verified by transformation of DH5α competent bacteria and single colony Sanger sequencing. At the same time, the control gene mCherry was connected to the pLenti lentiviral expression vector by the same method to construct the pLenti-mCherry control recombinant plasmid.

[0071] 2. Preparation of recombinant lentivirus packaging for overexpressing LSH

[0072] (1) Cultivation of lentiviral packaging cells 293FT cells

[0073] Human embryonic kidney 293FT cells were cultured in DMEM medium (Gibco, C11995) supplemented with 10% fetal bovine serum (BI) and 1% Penicillin-Streptomycin dual antibody (Sanggong). 293FT cells were placed in a 37°C cell culture incubator (containing 5% CO 2 ) and when the cells reached about 90% confluence, they were trypsinized and passaged at a ratio of 1:4 or 1:6.

[0074] (2) Packaging and preparation of recombinant lentivirus

[0075] The lentiviral recombinant expression plasmid pLenti-hLSH and lentiviral packaging plasmids (pMDLg / RRE plasmid, pVSVG plasmid and pRSV / Rev plasmid) were introduced into 293FT cells using the cationic non-liposomal transfection reagent VigoFect (Vigos Corporation). Fresh cell culture medium was replaced 6 hours after transfection to continue culture. After 48 hours and 72 hours of continuous culture, the cell culture medium containing lentivirus was collected, and the supernatant was obtained by centrifugation and filtered through a 0.45 μm syringe filter to obtain a lentiviral suspension.

[0076] 3. Establishment of IMR90 cell line with stable overexpression of LSH

[0077] (1) Cultivation of human embryonic fibroblast IMR90 cells

[0078] Human embryonic lung fibroblast IMR90 cells were cultured in MEM medium (Sigma, M4655) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Sangon), 1 mM sodium pyruvate (Gibco), 1% non-essential amino acids (Sigma) and 1% penicillin-streptomycin solution (Biyuntian). IMR90 cells were placed in a 37°C cell culture incubator (containing 5% CO 2 ) and when the cells reached about 90% confluence, they were trypsinized and passaged at a ratio of 1:4.

[0079] (2) Lentivirus infection of IMR90 cells

[0080] IMR90 cells with a lower passage number were subcultured into 3.5 cm culture dishes. After the cells were completely attached to the wall, the culture medium was discarded. 1 mL of filtered virus suspension and 1 mL of preheated fresh culture medium were mixed in equal proportions and added to the cells for infection. At the same time, 0.5 μL of 10 mg / mL Polybrene was added to improve the infection efficiency. After gently shaking to mix well, the cells were placed in a 37°C cell culture incubator (containing 5% CO 2) and continue to culture. After 24 hours of infection, the liquid in the infected cell culture dish was discarded, and 2 mL of a mixture of virus suspension and cell culture medium in equal proportions was added again for secondary infection. After 24 hours of secondary infection, the liquid in the infected cell culture dish was discarded, and 2 mL of fresh cell culture medium was added for continued culture.

[0081] (3) Screening of positive cells infected by lentivirus

[0082] On the 3rd or 4th day after lentivirus infection, positive cells are screened using the resistance gene or fluorescent protein carried on the pLenti recombinant plasmid integrated into the host cell genome. Specifically, resistance screening can be performed by adding corresponding antibiotics (such as Puromycin) to the culture medium of infected cells, and cells that survive after one week of continuous screening are cells that have been successfully infected by the lentivirus. Alternatively, cells that stably express the corresponding fluorescent protein can be separated by flow cytometry sorting, and these cells with positive fluorescent signals are cells that have been successfully infected.

[0083] (4) Identification of IMR90 cells stably overexpressing LSH

[0084] After screening the IMR90 cells that were successfully infected with the lentivirus, the expression of LSH in the positive cells needs to be further identified. Figure 1a It shows that during the replicative senescence of IMR90 fibroblasts, overexpression of LSH by lentiviral infection can effectively inhibit the loss of DNA methylation specific to heterochromatin regions; first, at the DNA level, the PCR method was used to confirm the insertion of exogenous LSH expression elements in the genome of positive cells. Secondly, at the RNA level, quantitative reverse transcription PCR (RT-qPCR) or transcriptome sequencing methods were used to identify the overexpression of LSH at the transcript level in positive cells (as shown in Figure 1a). Finally, at the protein level, the Western Blot method was used to determine the degree of LSH overexpression at the protein level ( Figure 1b As shown in Figure 2 ). At both the RNA and protein levels, LSH was significantly overexpressed by about several dozen times, indicating that LSH-overexpressing IMR90 cells mediated by lentiviral infection were successfully established.

[0085] 4. LSH overexpression delays loss of heterochromatin DNA methylation during long-term proliferation

[0086] Whole genome bisulfite sequencing (WGBS) was used to detect changes in DNA methylation levels in heterochromatin regions in IMR90 cells overexpressing mCherry and hLSH during replicative senescence caused by long-term continuous expansion. The specific experimental steps are as follows:

[0087] (1) Establishment of a replicative senescence cell model induced by long-term cell proliferation

[0088] IMR90 cells with a low number of passages (cell divisions of about 20 times, PD20) are continuously subcultured in vitro. After a long period of repeated expansion (about PD55-PD65), IMR90 cells will enter the cell cycle arrest stage and show phenotypes related to cell senescence. This process is the replicative senescence of IMR90 cells.

[0089] In young wild-type IMR90 cells that have not been cultured for long-term amplification, the mCherry overexpression control group and hLSH overexpression experimental group were established by lentiviral infection. The control group and experimental group cells were then cultured for a long time in parallel. After the cells entered the division arrest stage, the WGBS technique was used to detect the effect of LSH overexpression on the loss of heterochromatin DNA methylation caused by long-term cell proliferation.

[0090] (2) Extraction of genomic DNA with phenol-chloroform

[0091] 1) The IMR90 cells of the control group and the experimental group with lower and higher passage numbers were cultured in 6 cm cell culture dishes. When the cells grew to occupy about 90% of the bottom area of ​​the culture dish, they were digested with trypsin and collected into a new 1.5 mL centrifuge tube. After centrifugation at 900 rpm for 3 min, the supernatant was discarded and the cells were washed once with PBS.

[0092] 2) Resuspend the cell pellet washed with PBS in 1 mL of cell lysis buffer, add 30 μL of 10% SDS per mL of cell lysis buffer and mix thoroughly by pipetting. The solution should become viscous.

[0093] 3) Add 30 μL of 20 mg / mL RNase A and mix thoroughly by pipetting, then place in a 37°C water bath for digestion for about 3 hours;

[0094] 4) Add 20 μL of 20 mg / mL proteinase K and mix well by pipetting, then place in a 65°C water bath overnight;

[0095] 5) The next day, take the centrifuge tube out of the 65°C water bath and place it at room temperature for 5 minutes to allow it to cool back to room temperature;

[0096] 6) Add an equal amount of phenol-chloroform solution to the solution and vortex at maximum speed for 20 seconds to mix thoroughly;

[0097] 7) Centrifuge at 13.3 krpm for 15 minutes and transfer the upper aqueous phase containing DNA to a new 1.5 mL centrifuge tube;

[0098] 8) To obtain cleaner DNA, add an equal amount of phenol chloroform to the supernatant, shake well to mix, centrifuge at 13.3 krpm for 15 minutes, and transfer the upper aqueous phase to a new centrifuge tube;

[0099] 9) Add 1 / 10 volume of 4M NaCl solution to the supernatant, add 2.5 volumes of anhydrous ethanol, add 1 μL 10mg / ml glycogen, and mix thoroughly by inverting. If the amount of DNA is large enough, filamentous DNA precipitation can be observed at this time;

[0100] 10) Centrifuge at 13.3 krpm for 2 minutes, discard the supernatant, add 75% ethanol, mix thoroughly by inversion, centrifuge at 13.3 krpm for 2 minutes, and discard the supernatant;

[0101] 11) Add 75% ethanol again and mix by inversion. Centrifuge at 13.3 krpm for 2 minutes, discard the supernatant, and gently centrifuge the remaining liquid to the bottom of the tube in a centrifuge. After aspirating the remaining liquid, dry the precipitate thoroughly to evaporate the ethanol.

[0102] 12) Add 50 μL of 1×TE solution or 10 mM Tris-HCl (pH 8.0) solution to the precipitate to dissolve the DNA. Incubate the precipitate at room temperature for several hours or in a 4°C refrigerator overnight. After the DNA is completely dissolved, transfer it to -20°C for storage.

[0103] (3) Ultrasonic fragmentation of genomic DNA

[0104] 1) Take 2 μg of genomic DNA into a 200 μL centrifuge tube, add 10 μL of λ-DNA (1 ng / μg) as "Spike-in", then add appropriate amount of 1×TE to make up the solution to 125 μL, and transfer all to Covaris microTUBE (ultrasonic tube);

[0105] 2) Turn on the Covaris sonicator, place the sonication tube containing the DNA sample into the sonication tank, select the sonication program with a target fragment size of 200 bp, and run the program to break the genomic DNA into the target fragment size;

[0106] (4) Screening of genomic DNA fragment size

[0107] 1) Take 120 μL of the DNA solution after ultrasonic fragmentation into a 1.5 mL centrifuge tube, add 72 μL of AMPure magnetic beads (0.6×), mix thoroughly by pipetting, and place at room temperature (23°C) for 10 minutes;

[0108] 2) Place the centrifuge tube on the magnetic stand and let it stand for 5 minutes. At this time, the magnetic beads will gather on the tube wall on one side of the magnetic stand and the solution will become clear.

[0109] 3) Keep the centrifuge tube on the magnetic stand, transfer the supernatant to a new 1.5 mL centrifuge tube, and discard the magnetic bead precipitate;

[0110] 4) Place the new tube containing the supernatant on the magnetic stand for 5 minutes, and take 185 μL of the supernatant again into a new 1.5 mL centrifuge tube to fully remove the magnetic beads in the solution. At this time, DNA fragments larger than 600 bp are bound to the magnetic beads and discarded, while the supernatant contains DNA fragments smaller than 600 bp;

[0111] 5) Add 105 μL AMPure magnetic beads to the centrifuge tube, mix thoroughly by pipetting, and place at room temperature (23°C) for 10 minutes. At this time, the total volume of magnetic beads in the solution reaches 1.4× of the DNA solution, and the magnetic beads can bind to DNA fragments larger than 150 bp.

[0112] 6) Place the centrifuge tube on the magnetic stand for 5 minutes until the solution becomes clear, keep the centrifuge tube on the magnetic stand, and discard the supernatant (containing non-target DNA fragments less than 150 bp);

[0113] 7) Add 500 μL of 80% ethanol to the centrifuge tube, quickly rotate the centrifuge tube 180° on the magnetic rack and repeat 6-10 times, then let the centrifuge tube stand on the magnetic rack for 1 minute and discard the supernatant, repeat twice to fully wash the magnetic beads;

[0114] 8) Centrifuge the tube at 3,000g for 5 seconds to centrifuge the remaining ethanol to the bottom of the tube, discard the remaining liquid at the bottom of the tube, and keep the centrifuge tube lid open and let it stand at room temperature for several minutes to allow the residual ethanol to fully evaporate;

[0115] 9) When the magnetic bead precipitate is cracked, add 50 μL 10 mM Tris-HCl (pH 8.0), pipette up and down for more than 15 times, and let stand at room temperature for 10 minutes;

[0116] 10) Place the centrifuge tube on the magnetic stand for at least 2 minutes to allow the magnetic beads to fully aggregate, and transfer the supernatant to a new 1.5 mL centrifuge tube. Repeat twice to fully remove the magnetic beads in the solution.

[0117] 11) The DNA concentration was measured using a Qubit 2.0 fluorometer, and the measurement system is shown in Table 1 below:

[0118] Table 1

[0119] DNA 1μL Qubit dyes 1μL Buffer QB 198μL Total 200μL

[0120] 12) If the subsequent experimental operation is temporarily interrupted, the genomic DNA after fragment size screening can be stored at -20℃.

[0121] (5) Repair of genome fragment ends

[0122] 1) Take 500 ng of genomic DNA after fragment size screening, and use "KAPA HyperPrep Kits" (DNA library construction kit) to perform end-filling and A-tailing reaction, wherein the reaction solution preparation system is shown in Table 2 below:

[0123] Table 2

[0124] Genomic DNA fragments 500ng End Repair & A-Tailing Buffer 7μL End Repair&A-Tailing Enzyme Mix 3μL Ultrapure water Make up to 60 μL Total volume 60μL

[0125] 2) After the reaction system is fully mixed, the reaction is carried out on a PCR instrument according to the following procedures:

[0126] 20℃ 30min

[0127] 65℃ 30min

[0128] 4℃ ∞

[0129] The temperature of the heated cover needs to be set to 85°C, rather than the 105°C set for conventional PCR.

[0130] 3) After the reaction is completed, proceed to the next step of sequencing adapter ligation reaction immediately.

[0131] (6) Methylation sequencing adapter ligation

[0132] 1) Use "KAPA HyperPrep Kits" to connect the sequencing adapters with methylation modification to the two ends of the genomic DNA fragments after end filling and A tailing. The reaction system is shown in Table 3 below, and the preparation method is as follows:

[0133] Table 3

[0134]

[0135]

[0136] 2) After the reaction system is fully mixed, incubate at 20°C for 15 minutes.

[0137] (7) Purification of ligation reaction products

[0138] 1) Take 105 μL of the previous step ligation product into a 1.5 mL centrifuge tube, add 126 μL AMPure magnetic beads (1.2×), mix thoroughly by pipetting, and place at room temperature (23°C) for 10 minutes.

[0139] 2) Place the centrifuge tube on the magnetic stand for 5 minutes until the solution becomes clear, keep the centrifuge tube on the magnetic stand, and discard the supernatant;

[0140] 3) Add 500 μL of 80% ethanol to the centrifuge tube, quickly rotate the centrifuge tube 180° on the magnetic rack and repeat 6-10 times, then let the centrifuge tube stand on the magnetic rack for 1 minute and discard the supernatant, repeat twice to fully wash the magnetic beads;

[0141] 4) Centrifuge the tube at 3,000g for 5 seconds to centrifuge the remaining ethanol to the bottom of the tube, discard the remaining liquid at the bottom of the tube, and keep the centrifuge tube lid open and let it stand at room temperature for several minutes to allow the residual ethanol to fully evaporate;

[0142] 5) When the magnetic bead precipitate is cracked, add 40 μL 10 mM Tris-HCl (pH 8.0), pipette up and down for more than 15 times, and let stand at room temperature for 10 minutes;

[0143] 6) Place the centrifuge tube on the magnetic stand for at least 2 minutes to allow the magnetic beads to fully aggregate, and transfer the supernatant to a new 1.5 mL centrifuge tube. Repeat twice to fully remove the magnetic beads in the solution.

[0144] 7) The concentration of the purified DNA product was measured using a Qubit 2.0 fluorometer.

[0145] (8) Bisulfite conversion

[0146] 1) Perform bisulfite conversion on the purified ligation product using the EpiTect Bisulfite Kit. First, add 800 μL of ultrapure water to a tube of Bisulfite Mix powder, vortex thoroughly to mix to completely dissolve it, dispense into multiple 1.5 mL centrifuge tubes at a volume of 90 μL per tube, and store at -20°C for long-term storage.

[0147] 2) Prepare a bisulfite reaction system according to the following system, and each component needs to be added in the order of the following Table 4:

[0148] Table 4

[0149] Genomic DNA fragments 20μL Bisulfite Mix 85μL DNA Protect Buffer 35μL Total volume 140μL

[0150] 3) After the reaction system is fully mixed, the reaction is carried out on a PCR instrument according to the following procedures:

[0151]

[0152]

[0153] 4) After the bisulfite conversion reaction is completed, the reaction product is purified by column purification using the "EpiTect Bisulfite Kit", and finally the DNA is eluted using 20 μL of elution buffer EB.

[0154] (9) Low cycle PCR amplification

[0155] 1) PCR amplification of bisulfite-converted genomic DNA fragments was performed using "High Fidelity HotStart Uracil with ReadyMix" (usually referred to as HotStart Uracil+ReadyMix, a PCR premix reagent), wherein the reaction system is shown in Table 5 below, and the preparation method is as follows:

[0156] Table 5

[0157] Bisulfite converted DNA 10μL Primer (including i5 index) 3μL Primer (including i7 index) 3μL 2×KAPA Uracil Mix 25μL Ultrapure water 9μL Total volume 50μL

[0158] 2) After the reaction system is fully mixed, the reaction is carried out on a PCR instrument according to the following procedures:

[0159]

[0160] 30s-15s-1min, repeat 6 times.

[0161] 3) After the reaction is completed, the PCR product is transferred to a 1.5 mL centrifuge tube and purified by adding 50 μL AMPure magnetic beads (1×). Finally, 20 μL 10 mM Tris-HCl (pH 8.0) is used for elution to obtain the constructed DNA library.

[0162] 4) Use Qubit 2.0 Fluorometer to measure the concentration of the DNA library, and take an appropriate volume of the library and send it to a second-generation sequencing company for high-throughput sequencing.

[0163] (10) Bioinformatics analysis of WGBS sequencing data

[0164] 1) Use the software "FastQC" to perform quality detection on the original sequencing files in fastq format;

[0165] 2) Use the software “Trimmomatic” to remove sequencing adapter sequences and low-quality sequences in the original sequencing files;

[0166] 3) Use the software “Bismark Mapper” to align the raw sequencing files to the hg19 or hg38 human reference genome;

[0167] 4) Use the software “MethylDackel” to extract the DNA methylation level information of all CpG sites in the genome;

[0168] 5) The bed format file containing the methylation information of all CpG sites was converted into a bigwig file using the software “Wig / BedGraph-to-bigWig” and further visualized and analyzed in the IGV genome browser.

[0169] (11) Experimental results analysis

[0170] 1) Starting from the genome-wide DNA methylation level, see Figure 1c It can be found that compared with the IMR90 cells that were not cultured for a long time at the beginning (left column), the control cells with mCherry overexpression after long-term culture showed a significant decrease in the overall DNA methylation level (middle column), while this decrease was very effectively inhibited in the experimental group cells with LSH overexpression (right column);

[0171] 2) From the visualization results, see Figure 1d , it can be intuitively seen that compared with the young cells (P9) that were not cultured for a long time at the beginning, the control group cells (mCherry P19) had a significant loss of heterochromatin-specific methylation during the long-term continuous expansion process, while this loss was effectively inhibited in the experimental group cells (LSH P19) with overexpression of LSH;

[0172] 3) From the DNA methylation heat map, see Figure 1e Compared with young IMR90 cells that did not proliferate extensively at the beginning, the control group cells with mCherry overexpression showed a significant loss of DNA methylation over a large range after long-term subculture, while LSH overexpression could effectively delay this methylation loss.

[0173] 4) Specific analysis of DNA methylation changes in the early and late replication regions during long-term passage, see Figure 1f As shown in the figure, it can be seen that the inhibitory effect of LSH on DNA methylation loss is more obvious in the late region of DNA replication (ie, heterochromatin region).

[0174] All of the above results prove that LSH overexpression can indeed effectively inhibit the loss of heterochromatin DNA methylation that occurs in IMR90 fibroblasts during long-term sustained proliferation, providing a new and effective perspective and means for intervening in the epigenetic imbalance produced during cell therapy.

[0175] 5. LSH overexpression delays transposon activation and inflammatory response during long-term proliferation

[0176] RNA-seq transcriptome sequencing technology was used to detect the differences in gene expression between the mCherry overexpression control group cells and the hLSH overexpression experimental group cells before and after long-term subculture. The specific experimental steps are as follows:

[0177] (1) Total RNA extraction and sequencing:

[0178] 1) Take out IMR90 cells from the incubator, discard the culture medium, and add 1 mL of TRIzol directly to the culture dish. Use a pipette to repeatedly pipette up and down to mix, and let it stand for about 5 minutes to allow the cells to fully lyse;

[0179] 2) Add 0.2 mL of chloroform to 1 mL of TRIzol lysis buffer and vortex at maximum speed for 20 seconds. Mix thoroughly and incubate for 2-3 minutes.

[0180] 3) Centrifuge at 12,000 × g for 15 minutes at 4°C. The mixture can be separated into a lower organic phase, a middle phase, and an upper aqueous phase containing RNA.

[0181] 4) Transfer 0.4 mL of the upper aqueous phase containing RNA to a new 1.5 mL centrifuge tube, add 10 μg of RNase-free Glycogen and 0.5 mL of isopropanol to precipitate RNA, mix thoroughly and let stand at 4°C for 10 minutes, then centrifuge at 12,000 × g for 10 minutes in a 4°C centrifuge. After centrifugation, a white precipitate can be observed at the bottom of the tube;

[0182] 5) After carefully discarding the supernatant, wash the RNA precipitate twice with 75% ethanol, centrifuge at 7,500×g for 5 minutes at 4°C, discard the supernatant, and leave the tube open for 5-10 minutes to allow the ethanol to evaporate completely;

[0183] 6) Add 20-50 μL of RNase-free ultrapure water to dissolve the RNA precipitate. After it is fully dissolved, use Nanodrop ultra-micro spectrophotometer to measure the RNA concentration, and detect the OD values ​​at 260nm and 280nm wavelengths to evaluate the RNA purity and quality, and then send it to the second-generation sequencing company for RNA-seq library construction and sequencing.

[0184] (2) Bioinformatics analysis of RNA-seq transcriptome sequencing data

[0185] 1) Use the software "FastQC" to perform quality detection on the original sequencing files in fastq format;

[0186] 2) Use the software “Trim Galore!” to remove sequencing adapter sequences and low-quality sequences in the original sequencing files;

[0187] 3) Use the software “RNA STAR” to align the raw sequencing files to the hg19 or hg38 human reference genome;

[0188] 4) Use the software “featureCounts” to calculate the number of reads that can be mapped to each coding gene or transposon element;

[0189] 5) The software “DESeq2” was used to identify genes and transposon elements that were significantly differentially expressed during long-term sustained proliferation;

[0190] 6) Use the software “Volcano Plot” to draw a volcano plot of differentially expressed genes, and use the software “heatmap2” to draw a heat map of differentially expressed genes;

[0191] 7) The online tool “DAVID” was used to perform GO analysis on the differentially expressed genes to identify biological pathways with significant changes.

[0192] (3) Experimental results analysis

[0193] 1) By differentially analyzing the expression of transposon elements in IMR90 cells during long-term passage, see Figure 2a As shown in the figure, it can be found that after a large number of cell proliferations, a large number of abnormal activation of transposon elements exist on the genome, and the activation of most transposon elements can be significantly inhibited by LSH overexpression ( Figure 2b and Figure 2c ). Compared with transposon elements that cannot be inhibited by LSH overexpression, transposon elements that can be effectively inhibited by LSH overexpression will show a more dramatic decrease in DNA methylation during long-term continuous expansion, and this DNA methylation loss can be inhibited by LSH overexpression ( Figure 2d ), which indicates that LSH overexpression can prevent the abnormal activation of various transposon elements in the heterochromatin region by delaying the loss of DNA methylation in the heterochromatin region during long-term proliferation;

[0194] 2) According to the GO enrichment analysis results of differentially expressed genes, see Figure 2eAs shown in the figure, a large number of inflammatory response-related genes were significantly upregulated during the long-term expansion of IMR90 cells, and compared with the mCherry control group cells, this inflammatory response was effectively inhibited in the LSH overexpression experimental group. These inflammatory response-related genes come from multiple inflammatory signaling pathways, including type I interferon pathways (such as IFI6 / 27 / 30 / 44, ISG15, OAS1 / 2 / 3, GBP3 and DDX60L, etc.), aging-related secretory phenotype factors (SASP factors, such as IL-6 and MMP10, etc.), TNF signaling pathways (TRAF1, TNFRSF10A / 11B / 14 and BIRC3, etc.), nucleic acid sensing proteins (STING and RIG-I, etc.) and innate immune pathways (CD14, C6, PTX3 and NLRP10, etc.) ( Figure 2f ). These results fully demonstrate that LSH overexpression can inhibit the activation of transposon elements during long-term proliferation to prevent the activation of various cytoplasmic nucleic acid sensing proteins, thereby reducing the occurrence of chronic inflammatory responses.

[0195] 6. LSH overexpression improves senescence-related cell phenotypes caused by long-term proliferation

[0196] (1) LSH overexpression increases the proliferation capacity of senescent cells

[0197] IMR90 cells were subcultured continuously until they entered the replicative senescence stage. A small amount of cells were taken for counting using a cell counting plate at each subculture, and a fixed number of cells were seeded into a new cell culture dish for subculture. Based on the cell counting results at each subculture, the growth curves of the mCherry overexpression control group cells and the LSH overexpression experimental group cells were drawn. Figure 2g It can be found that LSH overexpression can significantly delay the cell cycle arrest phenotype during replicative senescence, allowing IMR90 cells to maintain a strong proliferation ability after undergoing a large amount of cell proliferation.

[0198] (2) LSH overexpression improves the enlarged phenotype of senescent cells

[0199] Flow cytometry was used to detect the size of senescent cells after long-term subculture, and the FSC intensity was used to measure the cell diameter. Figure 2h It can be found that the cells in the control group have a significant increase in cell volume during long-term continuous expansion, and LSH overexpression can significantly improve this cell enlargement phenotype associated with cell aging.

[0200] These results indicate that LSH overexpression can not only slow down the abnormal activation of transposon elements and the generation of chronic inflammatory responses during long-term proliferation by inhibiting the loss of heterochromatin DNA methylation, but can also further improve various cell senescence-related cell phenotypes such as slowed cell proliferation and increased cell volume. This provides a new and effective means to improve the long-term maintenance of cell function during cell therapy and enhance the clinical therapeutic effect of cell drugs.

[0201] Example 2

[0202] Overexpression of LSH can improve the proliferation ability and effector function of human T lymphocytes during long-term clonal expansion. The specific experimental steps are as follows:

[0203] 1. Preparation of recombinant lentivirus packaging for overexpressing LSH

[0204] (1) Construction of LSH overexpression recombinant lentiviral expression vector

[0205] The LSH coding sequence and the pCDH lentiviral vector were linearized by PCR, and homology arms were introduced at both ends of the fragment. Then, the LSH coding sequence was connected to the pCDH lentiviral expression vector by the Gibson Assembly method to construct the pCDH-LSH recombinant plasmid. The correctness of the recombinant plasmid was verified by transforming DH5α competent bacteria and single colony Sanger sequencing. At the same time, the control gene mCherry was connected to the pCDH lentiviral vector by the same method to construct the pCDH-mCherry control recombinant plasmid.

[0206] (2) Packaging and preparation of LSH overexpression recombinant lentivirus

[0207] 1) One day before transfection, subculture 293T cells into an appropriate number of 10 cm cell culture dishes, with approximately 4 × 10 cells per dish. 6 about;

[0208] 2) When the cell confluence reaches about 90%, the cells are transfected using the calcium phosphate transfection method, wherein the single-dish transfection system and configuration method are shown in Table 6 below:

[0209] Liquid A:

[0210] Table 6

[0211] pCDH Lentivirus Recombinant Plasmid 15 μg REV Lentiviral Packaging Plasmid 6μg RRE Lentiviral Packaging Plasmid 7.5 μg VSVG lentiviral packaging plasmid 6μg <![CDATA[2.5M CaCl 2 ]]> 50μL Sterile ultrapure water Make up to 500 μL Total volume 500μL

[0212] Solution B: 500 μL 2×HBS buffer

[0213] 3) After the preparation of solution A, let it stand at room temperature for 3 minutes, then add solution A to solution B, quickly pipette and mix 30-40 times to produce appropriate amount of bubbles. At this time, you can observe that the transfection mixture produces a slightly white precipitate (i.e. calcium transfer particles). Add the mixed liquid to 293T cells, shake gently and place in a 37°C cell culture incubator (containing 5% CO 2 ) for cultivation;

[0214] 4) 6-8 hours after transfection, discard the supernatant containing the transfection reagent and add 15 mL of fresh preheated cell culture medium to continue culturing;

[0215] 5) After 48 hours of replacing the culture medium, collect the cell supernatant containing the lentivirus, store it at 4°C, and add 10 mL of fresh preheated cell culture medium to continue culturing;

[0216] 6) After 72 hours of replacing the culture medium, the virus-containing supernatant was collected again and combined with the cell supernatant collected for the first time. The supernatant was centrifuged and filtered through a 0.45 μm syringe filter to obtain the lentiviral stock solution;

[0217] 7) The virus stock solution was centrifuged and concentrated 30 times using an ultrafiltration tube with a molecular weight cutoff of 100 kD. When the virus reached the corresponding volume, 10 mL of human T cell culture medium was added to resuspend it and centrifuged again to the target concentrated volume;

[0218] 8) In a biosafety cabinet, filter the concentrated virus suspension using a 0.22 μm syringe filter, divide into appropriate volumes, and freeze at -80°C.

[0219] 2. Lentivirus infection of human T cells to achieve stable overexpression of LSH

[0220] After T lymphocytes were isolated from human PBMCs, they were activated in vitro using αCD3 / αCD28 antibodies, and the LSH encoding gene was transferred into the T lymphocytes using lentiviral infection to detect the role of LSH overexpression in the long-term clonal expansion of T lymphocytes (see the experimental procedure for details). Figure 3a ), the specific experimental steps are as follows:

[0221] (1) Isolation of human peripheral blood mononuclear cells

[0222] 1) Dilute human peripheral blood according to a fixed ratio, taking 50 mL of concentrated human peripheral blood, adding 70 mL of blank RPMI 1640, and mixing the two thoroughly;

[0223] 2) Add 15 mL of Ficoll-Paque to a 50 mL centrifuge tube. TMLymphocyte separation fluid, and slowly add 30mL of diluted human peripheral blood along the wall of the centrifuge tube, then gently place the centrifuge tube in a horizontal centrifuge, adjust the centrifuge speed to 3 and 1, and centrifuge at 2,200rpm for 25min at 20℃;

[0224] 3) After centrifugation is completed, gently remove the centrifuge tube from the centrifuge. You can observe that the liquid in the centrifuge tube is stratified. From top to bottom, it is divided into four layers: plasma layer, mononuclear cell layer, lymphocyte separation fluid layer and granulocyte-erythrocyte layer. Slowly aspirate the top layer of plasma, then slowly aspirate the second layer of mononuclear lymphocyte layer and transfer it to a 50mL centrifuge tube;

[0225] 4) Add serum-free RPMI 1640 medium to the liquid containing monocytes to make up to 50 mL, mix thoroughly, and centrifuge at 1,800 rpm for 10 min at 20°C, with both the acceleration and deceleration speeds set to 9;

[0226] 5) After centrifugation, cell pellets can be observed at the bottom of the centrifuge tube. Carefully discard the supernatant and resuspend the cells in serum-free RPMI 1640 medium;

[0227] 6) Centrifuge at 1,600 rpm for 6 min at 20°C, and resuspend the cell pellet in serum-free RPMI 1640 medium;

[0228] 7) Take a small amount of cells and count them using a cell counting plate, add trypan blue to analyze cell viability, and then divide the cells into appropriate amounts and freeze them.

[0229] (2) In vitro activation of T cells in human peripheral blood

[0230] 1) Take out the frozen human peripheral blood mononuclear cells (PBMC) from liquid nitrogen, thaw quickly in a 37°C water bath, count the cells using trypan blue, and adjust the cell density to 1×10 7 The cells were seeded in 24-well plates and placed in a 37°C cell culture incubator (containing 5% CO 2 ) for cultivation;

[0231] 2) After culturing the revived PBMC overnight, the cells were counted again using trypan blue, and the required volume of αCD3 / αCD28 magnetic beads (Gibco, 40203D) was calculated based on the number of cells, where 4×10 5 Cells require 1 μL of magnetic beads for activation;

[0232] 3) Wash the αCD3 / αCD28 magnetic beads with culture medium. First, add 1 mL of blank RPMI 1640 culture medium to the sterile flow tube, then add the corresponding volume of magnetic beads, gently pipette to mix, and then place the sterile flow tube on the magnetic rack for about half a minute. After the magnetic beads gather on the side close to the tube wall of the magnetic rack, aspirate and discard the supernatant of the culture medium;

[0233] 4) Add PBMC cell suspension to the magnetic bead pellet, gently pipette to mix, place on a rotary mixer and incubate for 0.5 h, then transfer the cells to a 24-well plate and place in a 37°C cell culture incubator (containing 5% CO 2 ) and cultured overnight.

[0234] (3) Lentivirus infection of human T cells

[0235] 1) After 24 hours of activation, dilute the T cells to a density of 5×10 5 / mL, and 500 μL of cell suspension was added to a 24-well plate;

[0236] 2) Take the frozen lentivirus out of the -80°C freezer, slowly thaw on ice and return to room temperature, add 500 μL of virus suspension to each well and mix with the cell suspension in a 1:1 ratio, add Polybrene to promote infection (final concentration of 8 μg / mL), and add 100 U of cytokine IL-2 and mix thoroughly;

[0237] 3) Seal the 24-well plate for culturing T cells with plastic wrap and centrifuge at 2,000×g for 90 min in a centrifuge, with the centrifuge temperature set to 30°C, the speed of increase set to 3, and the speed of decrease set to 1. After centrifugation, take out the plate and place it in a 37°C cell culture incubator (containing 5% CO 2 ) for incubation and infection;

[0238] 4) After 4-6 hours of centrifugal infection, add 1 mL of cell culture medium to each well, mix thoroughly, and return to the 37°C cell culture incubator (containing 5% CO 2 ) to continue to cultivate;

[0239] 5) On the second day of lentiviral infection, carefully remove 1500 μL of supernatant from the cell culture wells, add 500 μL of lentiviral suspension again, add Polybrene and IL-2, and then perform a second centrifugation infection. 4-6 hours after the infection, add 1 mL of cell culture medium, and place in a 37°C cell culture incubator (containing 5% CO 2 ) to continue to cultivate;

[0240] 6) On the third day, discard the virus-containing cell supernatant, add 2 mL of pre-warmed fresh T cell culture medium, and add IL-2 (100 U) to continue culturing;

[0241] 7) On the 5th day, the fluorescent protein carried by the exogenous expression gene was used for flow cytometry analysis to determine the infection efficiency of the lentivirus, and the successfully infected T cells were sorted out by flow cytometry for further culture;

[0242] (4) Western Blot detection of overexpression efficiency

[0243] 1) Cell collection: Human T cells cultured in vitro were collected into centrifuge tubes and centrifuged at 1,800 rpm for 10 min. The supernatant was discarded and resuspended in 1 mL 1× PBS. After counting the cells using trypan blue, the cells were centrifuged again and the supernatant was discarded. 1×10 6 The cells were resuspended in 100 μL 1× PBS, and 100 μL 2× SDS loading buffer was added. After thorough mixing, the cells were heated and lysed in a 100°C metal bath for 10 min. The protein lysate was then centrifuged at 13,300 rpm for 5 min. The protein supernatant was transferred to a new centrifuge tube, and the cell residue precipitate was discarded. Finally, the protein sample was placed in a -20°C refrigerator for long-term storage.

[0244] 2) SDS-PAGE gel electrophoresis: Take the frozen protein samples out of the -20℃ refrigerator and heat them in a 100℃ metal bath for 5 minutes. After the samples are cooled, load them into the SDS-PAGE gel wells in a certain order. Pour an appropriate amount of 1× electrophoresis buffer into the electrophoresis tank and perform constant current electrophoresis at 20mA for 1.5 hours.

[0245] 3) Protein transfer: Cut a PVDF membrane of appropriate size and soak it in methanol for activation for 1 min. Place a sponge, four layers of filter paper, SDS-PAGE gel, PVDF membrane, four layers of filter paper and sponge on the transfer clip in order. Then put the transfer clip into the transfer tank, making sure that the side where the gel is placed faces the negative electrode. Pour an appropriate amount of 1× transfer buffer. After turning on the power, transfer the membrane at a constant current of 200 mA for 2 h to transfer the protein bands from the gel to the PVDF membrane.

[0246] 4) Blocking: After the transfer, remove the PVDF membrane and place it in a blocking solution containing 5% skimmed milk powder (prepared by TBST), and slowly shake it on a shaker at room temperature for 1-2 hours;

[0247] 5) Primary antibody incubation: After blocking, cut the target band of the corresponding molecular weight, put it into the primary antibody appropriately diluted in the blocking solution, and incubate it on a shaker at 4°C overnight;

[0248] 6) Washing the membrane: After the primary antibody incubation, immerse the PVDF membrane in TBST and wash it on a shaker at room temperature for 3 times, each time for 10 minutes;

[0249] 7) Secondary antibody incubation: Place the washed PVDF membrane in the secondary antibody appropriately diluted with TBST and incubate on a shaker at room temperature for 1-2 hours;

[0250] 8) Washing the membrane: After the secondary antibody incubation, immerse the PVDF membrane in TBST and wash it on a shaker at room temperature for 3 times, 10 minutes each time;

[0251] 9) Protein signal detection: Dip the fully washed PVDF membrane on absorbent paper and flatten it on a plastic film, evenly add chemiluminescent HRP substrate (A solution and B solution mixed in a ratio of 1:1) to the surface of the PVDF membrane, and after a suitable reaction time, put it into a dark box for pressing, and then develop it in a dark room using an automatic X-ray film developer;

[0252] 10) Test results: Figure 3b and Figure 3c The figure shows the changes in the expression levels of LSH protein in wild-type and LSH-overexpressing human T lymphocytes. Specifically, by detecting the expression of endogenous LSH protein in wild-type T lymphocytes, it can be found that the expression level of LSH gradually decreases with the extension of in vitro culture time ( Figure 3b ). In T lymphocytes infected with LSH overexpressing lentivirus, protein immunoblotting detection using antibodies targeting the HA tag carried by the exogenous LSH expression element can reveal that exogenous LSH protein is indeed successfully expressed in human T cells, proving that the lentivirus infection successfully introduced the exogenous LSH expression element into human T cells. At the same time, protein immunoblotting detection using antibodies targeting the C-terminal sequence of LSH itself can observe that compared with human T cells infected with mCherry control lentivirus, human T cells infected with LSH lentivirus have obvious LSH overexpression ( Figure 3c ), demonstrating the success of LSH overexpression mediated by lentiviral infection.

[0253] 3. LSH overexpression improves T cell function degradation during long-term clonal expansion

[0254] (1) LSH overexpression inhibits DNA methylation loss in heterochromatin regions of T cells

[0255] The WGBS technique was used to detect changes in DNA methylation levels in human T lymphocytes during long-term in vitro clonal expansion (see Figure 3d ), we found that LSH overexpression can effectively delay the decrease of DNA methylation level in heterochromatin regions during long-term proliferation, indicating that LSH overexpression in human T lymphocytes can indeed significantly improve the efficiency of maintaining DNA methylation in heterochromatin regions, thereby preventing the imbalance of epigenetic information in T cells during long-term continuous proliferation.

[0256] (2) LSH overexpression promotes the maintenance of effector function of T cells during long-term expansion

[0257] RNA-seq sequencing was used to detect the changes in the transcriptome of human T lymphocytes during long-term in vitro clonal expansion (see Figure 3e and 3f ), we found that LSH overexpression can significantly enhance the effective maintenance of T cell effector function during long-term continuous proliferation. By comparing and analyzing the differentially expressed genes in the control group with 4RFP overexpression and the experimental group with hLSH overexpression after large-scale clonal expansion, we can observe that the expression of a large number of T cell effector function-related genes is significantly increased in LSH overexpressing cells (see Figure 3e ), including effector function-related cytokines (such as IL-13, CSF1 / 2, CCL4 and IFNG, etc.), cell killing function-related proteins (such as GZMA, GZMB and FASLG, etc.), effector cell surface receptors (such as IL4R and IL2RA, etc.) and effector cell differentiation-related transcription factors (such as JAK3-STAT3, BATF and BHLHE40, etc.). At the same time, the results of GSEA analysis showed (such as Figure 3f ), the expression of genes related to T cell effector function will decrease significantly during long-term continuous expansion in vitro, and LSH overexpression can effectively delay the degradation of this T cell effector function. This shows that LSH overexpression can promote the long-term effective maintenance of T cell effector function by inhibiting the imbalance of epigenetic information of T lymphocytes in long-term clonal expansion.

[0258] (3) LSH overexpression improves the proliferation capacity of T cells during long-term clonal expansion

[0259] The human T cells in the mCherry overexpression control group and the LSH overexpression experimental group were cultured in vitro for a long time, and the number of T cells growing was counted every day during the culture process to draw the growth curve of the T cells in the control group and the experimental group (such as Figure 3g ). From the growth curve, it can be observed that in the long-term in vitro culture process, the experimental group cells with LSH overexpression showed a significant growth advantage compared with the control group cells. At the same time, the level of Ki-67, a proliferation-related marker in T lymphocytes, was detected by flow cytometry (such as Figure 3h ), it was found that the Ki-67 level in the LSH-overexpressing experimental group was significantly higher than that in the control group, and this increase was found in CD8 + Therefore, these results indicate that LSH overexpression can enhance T lymphocytes (especially CD8 + The cell proliferation capacity of T lymphocytes during long-term and large-scale expansion.

[0260] (4) LSH overexpression slows down T cell death in the late stage of clonal expansion

[0261] Since the T cells in the LSH overexpression experimental group showed a significant survival advantage in the late stage of in vitro proliferation, we further tested the survival of T cells in the late stage of proliferation. The dead cell fluorescent dye (A780) and flow cytometry were used to detect the changes in the live / dead cell ratio in the late stage of cell clone expansion (such as Figure 3i ), we were able to observe that with the extension of culture time, the proportion of live cells in the control group continued to decrease significantly, while LSH overexpression could effectively inhibit the gradual increase in the proportion of cell death, thereby significantly improving the cell survival ability of late clonal expansion T cells.

[0262] (5) LSH overexpression delays CD27 downregulation in long-term clonal expansion of T cells

[0263] Since the downregulation of the levels of co-stimulatory molecules CD27 and CD28 is a common cellular phenotype during T cell aging in vivo and long-term expansion in vitro, and CD27 and CD28 co-stimulatory molecules play an important role in the effective response of T cells to antigen stimulation and the exertion of effector functions, we further used flow cytometry to detect the changes in the expression of surface CD27 molecules in the long-term clonal expansion of T lymphocytes. The results showed that the proportion of CD27-positive cells decreased significantly during the long-term proliferation of the control group cells, and LSH overexpression can effectively prevent the downregulation of the CD27 molecule level. This shows that LSH overexpression can effectively improve the decrease in the expression level of co-stimulatory molecules in T lymphocytes during long-term clonal expansion, and improve the response ability of T cells to antigen stimulation.

[0264] These results indicate that LSH overexpression can effectively delay the loss of DNA methylation in heterochromatin regions during long-term clonal expansion of T lymphocytes, and significantly enhance the cell division and survival abilities of T cells in the late stage of sustained proliferation. At the same time, LSH overexpression can also effectively promote the long-term maintenance of T lymphocyte antigen response and effector functions, which provides a new optimization strategy for improving the efficacy of adoptive immune cell therapies such as CAR-T.

[0265] The above-mentioned relevant experimental research results of the present invention show that LSH overexpression can effectively inhibit the loss of DNA methylation in the heterochromatin region of human embryonic lung IMR90 fibroblasts during long-term continuous proliferation, so as to prevent abnormal activation of transposon elements in this genomic region, thereby reducing the generation of chronic inflammatory response, and significantly improving multiple cell phenotypes related to replicative aging such as cell cycle arrest and cell volume enlargement. At the same time, LSH overexpression can effectively improve the stability of heterochromatin in human T lymphocytes during long-term clonal expansion, thereby significantly improving the expansion ability and survival ability of T cells in the late stage of continuous proliferation, and promoting the long-term effective maintenance of T cell effector function.

[0266] The innovative research results of the present invention have strongly proved that the insufficient DNA methylation maintenance efficiency caused by the dense chromatin environment in the heterochromatin region is the reason for the continuous loss of DNA methylation when the cells continue to expand, and the overexpression of LSH can effectively improve the DNA methylation maintenance efficiency in the heterochromatin region to prevent the loss of DNA methylation, thereby improving a number of phenotypic defects caused by continuous cell expansion, such as the decline in cell proliferation ability, the upregulation of inflammatory response gene expression, and the degradation of immune cell effector function. This provides a new perspective for understanding epigenetic imbalance and cell function degradation during cell expansion, and provides a new strategy for improving the effective maintenance of cell function in cell adoptive therapy, and has a wide range of application value in improving the efficacy of various types of cell therapy. Therefore, the present invention provides a new research perspective and intervention means for effectively improving the stability of heterochromatin and enhancing the efficacy of cell therapy, and has a wide range of value in the clinical application of various cell drugs.

[0267] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Use of substances that increase LSH content / activity in the preparation of drugs / reagents for improving heterochromatin stability.

2. The use according to claim 1, characterized in that The substance that increases the expression level of LSH is an LSH overexpression substance that inhibits the loss of DNA methylation in heterochromatin regions.

3. The use according to claim 2, characterized in that The heterochromatin region is a chromatin region with histone H3K9me3 modification, HP1 protein or Lamin B1 protein highly enriched, or a DNA late replication region in the S phase of the cell cycle.

4. Application of substances that increase LSH content / activity in the preparation of drugs / reagents for enhancing the efficacy of cell therapy.

5. The use according to claim 4, characterized in that The medicine is a medicine that promotes the effective maintenance of cell function during cell therapy.

6. The use according to claim 5, characterized in that The cell therapy includes functional cell transplantation therapy and immune cell adoptive therapy.

7. The use according to claim 1 or 4, characterized in that: The substance for increasing the expression amount / activity of LSH is a LSH overexpression recombinant vector, a LSH overexpression recombinant lentivirus or a LSH overexpression recombinant cell.

8. The use according to claim 7, characterized in that The LSH overexpression recombinant vector is obtained by connecting the LSH gene coding sequence to a virus vector.

9. The use according to claim 8, characterized in that The LSH gene coding sequence is the original CDS coding sequence of the LSH gene, or the CDS coding sequence of the LSH gene after codon optimization, or the CDS coding sequence of the LSH gene after base sequence modification without changing the biological function of LSH itself.

10. The use according to claim 8, characterized in that The viral vector is a lentiviral vector, a retroviral vector or an AAV viral vector.

11. The use according to claim 8, characterized in that The LSH overexpression recombinant lentivirus is produced by co-transfecting the LSH overexpression recombinant vector and the virus packaging plasmid into human embryonic kidney HEK293T cells.

12. The use according to claim 11, characterized in that The virus packaging plasmids are pMDLg / pRRE, pRSV / REV and pVSVG.

13. The use according to claim 11, characterized in that The LSH overexpression recombinant cells are obtained by infecting host cells with LSH overexpression recombinant viruses.

14. The use according to claim 13, characterized in that The host cell is human embryonic lung IMR90 fibroblast, or human peripheral blood derived T lymphocyte, or other cell types used for cell therapy.

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